Alternaria redefined - CBS - KNAW

Woudenberg et al.
hyphomycetes, further complicating the taxonomic resolution in
this group of fungi. Several re-descriptions and revised criteria of
these genera (Saccardo 1886, Elliot 1917, Wiltshire 1933, 1938,
Joly 1964) resulted in a growing number of new species. Results of
a lifetime study on Alternaria taxonomy based upon morphological
characteristics were summarised in Simmons (2007), in which 275
Alternaria species were recognised. One species was transferred
to the genus Prathoda and three new genera, Alternariaster,
Chalastospora and Teretispora, were segregated from Alternaria.
Molecular studies revealed multiple non-monophyletic genera
within the Alternaria complex and Alternaria species clades,
which do not always correlate to species-groups based upon
morphological characteristics (Pryor & Gilbertson 2000, Chou &
Wu 2002, de Hoog & Horré 2002, Pryor & Bigelow 2003, Hong
et al. 2005, Inderbitzin et al. 2006, Pryor et al. 2009, Runa et al.
2009, Wang et al. 2011, Lawrence et al. 2012). The A. alternata,
A. brassicicola, A. infectoria, A. porri and A. radicina speciesgroups
were strongly supported by these studies and two new
species-groups, A. sonchi (Hong et al. 2005) and A. alternantherae
(Lawrence et al. 2012) and three new genera, Crivellia (Inderbitzin
et al. 2006), Undifilum (Pryor et al. 2009) and Sinomyces (Wang et
al. 2011), were described. The latest molecular revision of Alternaria
(Lawrence et al. 2013) introduced two new species groups, A.
panax and A. gypsophilae, and elevated eight species-groups
to sections within Alternaria. The sexual phylogenetic Alternaria
lineage, the A. infectoria species-group, did not get the status of
section, in contrast to the eight asexual phylogenetic lineages in
Alternaria. The Alternaria complex currently comprises the genera
Alternaria, Chalastospora (Simmons 2007), Crivellia, Embellisia,
Nimbya, Stemphylium, Ulocladium, Undifilum and the recently
described Sinomyces together with eight sections of Alternaria and
the A. infectoria species-group.
The aim of the present study was to delineate the phylogenetic
lineages within Alternaria and allied genera, and to create a robust
taxonomy. Phylogenetic inferences were conducted on sequence
data of parts of the 18S nrDNA (SSU), 28S nrDNA (LSU), the
internal transcribed spacer regions 1 and 2 and intervening
5.8S nrDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), RNA polymerase second largest subunit (RPB2) and
translation elongation factor 1-alpha (TEF1) gene regions of extype
and reference strains of Alternaria species and all available
allied genera.
MATERIAL AND METHODS
Isolates
Based on the ITS sequences of all ex-type or representative
strains from the Alternaria identification manual present at the
CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The
Netherlands (data not shown), 66 Alternaria strains were included
in this study together with 61 ex-type or representative strains of 16
related genera (Table 1). Alternaria is represented by the ex-type
or representative strains of the seven species-groups and species
that clustered outside known Alternaria clades. Because of the
size and complexity of the A. alternata, A. infectoria and A. porri
species-groups, we only included known species; the complete
species-groups will be treated in future studies.
Freeze-dried strains were revived in 2 mL malt/peptone (50 %
/ 50 %) and subsequently transferred to oatmeal agar (OA) (Crous
et al. 2009a). Strains of the CBS collection stored in liquid nitrogen
were transferred to OA directly from -80 ºC. DNA extraction was
performed using the UltraClean Microbial DNA Isolation Kit (MoBio
laboratories, Carlsbad, CA, USA), according to the manufacturer’s
instructions.
Taxonomy
Morphological descriptions were made for isolates grown on
synthetic nutrient-poor agar plates (SNA, Nirenberg 1976) with a
small piece of autoclaved filter paper placed onto the agar surface.
Cultures were incubated at moderate temperatures (~ 22 ºC)
under CoolWhite fluorescent light with an 8 h photoperiod for 7 d.
The sellotape technique was used for making slide preparations
(Crous et al. 2009a) with Shear’s medium as mounting fluid.
Photographs of characteristic structures were made with a Nikon
Eclipse 80i microscope using differential interference contrast
(DIC) illumination. Growth rates were measured after 5 and 7 d.
Colony characters were noted after 7 d, colony colours were rated
according to Rayner (1970). Nomenclatural data were deposited in
MycoBank (Crous et al. 2004).
PCR and sequencing
The SSU region was amplified with the primers NS1 and NS4 (White
et al. 1990), the LSU region with LSU1Fd (Crous et al. 2009b) and
LR5 (Vilgalys & Hester 1990), the ITS region with V9G (De Hoog
& Gerrits van den Ende 1998) and ITS4 (White et al. 1990), the
GAPDH region with gpd1 and gpd2 (Berbee et al. 1999), the RPB2
region with RPB2–5F2 (Sung et al. 2007) and fRPB2–7cR (Liu et
al. 1999) and the TEF1 gene with the primers EF1-728F and EF1-
986R (Carbone & Kohn 1999) or EF2 (O’Donnell et al. 1998). The
PCRs were performed in a MyCycler TM Thermal Cycler (Bio-Rad
Laboratories B.V., Veenendaal, The Netherlands) in a total volume
of 12.5 µL. The SSU and LSU PCR mixtures consisted of 1 µL
genomic DNA, 1´ GoTaq® Flexi buffer (Promega, Madison, WI,
USA), 2 µM MgCl 2
, 40 µM of each dNTP, 0.2 µM of each primer
and 0.25 Unit GoTaq® Flexi DNA polymerase (Promega). The
ITS and GAPDH PCR mixtures differed from the original mix by
containing 1 µM MgCl 2
, the RPB2 and TEF1 PCR mixtures differed
from the original mix by containing 2 µL genomic DNA and the
RPB2 mixture differed from the original mix by containing 0.5 U
instead of 0.25 U GoTaq® Flexi DNA polymerase. Conditions for
PCR amplification consisted of an initial denaturation step of 5 min
at 94 ºC followed by 35 cycles of 30 s at 94 ºC, 30 s at 48 ºC and
90 s at 72 ºC for SSU, LSU, ITS and 40 cycles of 30 s at 94 ºC, 30
s at 52 ºC / 59 ºC and 45 s at 72 ºC for TEF1 using respectively
EF2 or EF1-986R as reverse primer and a final elongation step of
7 min at 72 ºC. The partial RPB2 gene was obtained by using a
touchdown PCR protocol of 5 cycles of 45 s at 94 ºC, 45 s at 60
ºC and 2 min at 72 ºC, followed by 5 cycles with a 58 ºC annealing
temperature and 30 cycles with a 54 ºC annealing temperature.
The PCR products were sequenced in both directions using the
PCR primers and the BigDye Terminator v. 3.1 Cycle Sequencing
Kit (Applied Biosystems, Foster City, CA, USA), according to
the manufacturer’s recommendations, and analysed with an ABI
Prism 3730XL Sequencer (Applied Biosystems) according to the
manufacturer’s instructions. Consensus sequences were computed
from forward and reverse sequences using the BioNumerics v. 4.61
software package (Applied Maths, St-Martens-Latem, Belgium). All
generated sequences were deposited in GenBank (Table 1).
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